STRUCTURE OF INTEGRIN ALPHA V-beta 3 EXTRACELLULAR DOMAIN COMPLEXED WITH LIGAND

Abstract

The present invention relates to structure-based methods for identifying molecules which will bind to the αA-lacking integrin αVβ3 receptor extracellular domain and modulate function, e.g., by acting as receptor agonists, such as agonists that induce some or all of the biological responses induced by the ligand Arg-Gly-Asp-(D-Phe)-(N-methyl-Val), although the methods are not limited to the identification of this particular class of agonist. The invention also provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by structure coordinates of Table 1 or Table 2. In addition, the invention provides a crystal comprising an integrin IVJ3 extracellular domain alone or complexed with a cyclic RGD peptide as defined by the structural coordinates of Table 1 or Table 2.

1 . A method for determining whether a test compound is a potential modulator of αVβ3 integrin, the method comprising: 
 a) providing a computer model of the three-dimensional structure comprising a binding site of αVβ3 integrin defined by the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Tyr122; β3:Arg214; β3:Asn215; and β3:Arg216 according to Table 2;    b) providing a computer model of the three dimensional structure of a test compound;    c) computationally performing a fitting operation between the computer model of the binding site and the computer model of the test compound; and    d) evaluating the results of the fitting operation to evaluate the ability of the test compound to bind αVβ3 integrin;    wherein a test compound having the ability to bind αVβ3 integrin is a potential modulator of αVβ3 integrin.    
     
     
         2 . The method of  claim 1 , wherein the computer model of the three-dimensional structure of a test compound is from a database of compounds of known structure.  
     
     
         3 . The method of  claim 1 , wherein the three-dimensional structure of the binding site of αVβ3 integrin is defined by the atomic coordinates of αVβ3 integrin amino acids according to Table 2.  
     
     
         4 . The method of  claim 1 , wherein the fitting operation comprises determining an energy minima configuration of computer model of the three-dimensional structure of the test compound in the computer model of the three-dimensional structure of αVβ3 integrin.  
     
     
         5 . A method for determining whether a test compound is a potential modulator of αVβ3 integrin, the method comprising: 
 a) providing a computer model of the three-dimensional structure comprising a binding site of αVβ3 integrin defined by the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Asp119; β3:Ser121; β3:Tyr122; β3:Ser123; β3:Asp126; β3:Asp127; β3:Asp158; β3:Arg214; β3:Asn215; β3:Arg216; β3:Asp217; β3:Ala218; β3:Pro219; β3:Glu220; and β3:Asp251 according to Table 2;    b) providing a computer model of the three dimensional structure of a test compound;    c) computationally performing a fitting operation between the computer model of the binding site and the computer model of the test compound; and    d) evaluating the results of the fitting operation to evaluate the ability of the test compound to bind αVβ3 integrin;    wherein a test compound having the ability to bind αVβ3 integrin is a potential modulator of αVβ3 integrin.    
     
     
         6 . The method of  claim 5 , wherein the computer model of the three-dimensional structure of a binding site of αVβ3 is further defined by the inclusion of the atomic coordinates of one or more divalent cations according to Table 2.  
     
     
         7 . The method of  claim 5 , wherein the computer model of the three-dimensional structure of a test compound is from a database of compounds of known structure.  
     
     
         8 . The method of  claim 5 , wherein the three-dimensional structure of the binding site of αVβ3 integrin is defined by the atomic coordinates of αVβ3 integrin amino acids according to Table 2.  
     
     
         9 . The method of  claim 5 , wherein the fitting operation comprises determining an energy minima configuration of computer model of the three-dimensional structure of the test compound in the computer model of the three-dimensional structure of αVβ3.  
     
     
         10 . A method for determining whether a test compound is a potential modulator of αVβ3 integrin, the method comprising: 
 a) providing a computer model of the three-dimensional structure comprising a binding site of αVβ3 defined by the atomic coordinates of αVβ3 amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Asp119; β3:Ser121; β3:Tyr122; β3:Ser123; β3:Asp126; β3:Asp127; β3:Asp158; β3:Arg214; β3:Asn215; β3:Arg216; β3:Asp217; β3:Ala218; β3:Pro219; β3:Glu220; and β3:Asp251 according to Table 2;    b) providing a computer model of the three dimensional structure of a test compound;    c) computationally performing a fitting operation between the computer model of the binding site and the computer model of the test compound;    d) evaluating the results of the fitting operation to evaluate the ability of the test compound to bind αVβ3 integrin;    e) electing a test compound having the ability to bind αVβ3 integrin as a potential modulator of αVβ3 integrin;    f) obtaining or synthesizing the potential modulator; and    g) evaluating the ability of the potential modulator to modulate the activity of αVβ3 integrin.    
     
     
         11 . The method of  claim 5 , wherein the computer model of the three-dimensional structure of a binding site of αVβ3 integrin is further defined by the inclusion of the atomic coordinates of one or more divalent cations according to Table 2.  
     
     
         12 . The method of  claim 10 , wherein the fitting operation comprises determining an energy minima configuration of computer model of the three-dimensional structure of the test compound in the computer model of the three-dimensional structure of αVβ3 integrin.  
     
     
         13 . The method of  claim 10 , wherein the evaluating comprises determining the binding affinity of the test compound for αVβ3 integrin.  
     
     
         14 . A method for determining whether a test compound is a potential modulator of αVβ3 integrin, the method comprising: 
 a) providing a computer model of the three-dimensional structure of cyclo(RGDf-N-Me-V) according to Table 1 or Table 2;    b) providing a computer model of the three dimensional structure of a test compound;    c) computationally comparing the computer model of the binding site and the computer model of the test compound; and    d) evaluating the results of the comparison to evaluate the ability of the test compound to bind αVβ3 integrin;    wherein a test compound having a structure similar to cyclo(RGDf-N-Me-V) is a potential modulator of αVβ3 integrin.    
     
     
         15 . A method for determining whether a test compound is a potential modulator of αVβ3 integrin, the method comprising: 
 a) providing a computer model of the three-dimensional structure comprising an active site groove of αVβ3 integrin defined by the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Tyr122; β3:Arg214; β3:Asn215; and β3:Arg216 according to Table 2;    b) providing a computer model of the three dimensional structure of a test compound;    c) computationally performing a fitting operation between the computer model of the active site groove and the computer model of the test compound; and    d) evaluating the results of the fitting operation to evaluate the ability of the test compound to interact with the active site groove of αVβ3 integrin;    wherein a test compound having the ability to interact with the active site groove of αVβ3 integrin is a potential modulator of αVβ3 integrin.    
     
     
         16 . The method of  claim 15 , wherein the computer model of the three-dimensional structure of a test compound is from a database of compounds of known structure.  
     
     
         17 . The method of  claim 15 , wherein the three-dimensional structure of the active site groove of αVβ3 integrin is defined by the atomic coordinates of αVβ3 integrin amino acids according to Table 2.  
     
     
         18 . The method of  claim 15 , wherein the fitting operation comprises determining an energy minima configuration of computer model of the three-dimensional structure of the test compound in the computer model of the three-dimensional structure of αVβ3 integrin.  
     
     
         19 . The method according to  claim 15  wherein the active site groove is formed by the D3-A3, A3-B3, and D4-A4 loops (as shown in  FIG. 2A ).  
     
     
         20 . A method for evaluating the potential of a chemical entity to associate with: 
 a) a molecule or molecular complex comprising a binding pocket defined by the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Tyr122; β3:Arg214; β3:Asn215; and β3:Arg216 according to Table 2, or    b) a homologue of the molecule or molecular complex, wherein the homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 Å, the method comprising:    i) employing computational means to perform a fitting operation between the chemical entity and a binding pocket defined by the structure coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Tyr122; β3:Arg214; β3:Asn215; and β3:Arg216 according to Table 2 ±a root mean square deviation from the backbone of the amino acids of not more than 1.5 Å; and    ii) analyzing the results of the fitting operation to quantify the association between the chemical entity and the binding pocket.    
     
     
         21 . The method of  claim 20 , wherein the method evaluates the potential of a chemical entity to associate with: 
 a) a molecule or molecular complex comprising a binding pocket defined by the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Asp119; β3:Ser121; β3:Tyr122; β3:Ser123; β3:Asp126; β3:Asp127; β3:Asp158; β3:Arg214; β3:Asn215; β3:Arg216; β3:Asp217; β3:Ala218; β3:Pro219; β3:Glu220; and β3:Asp251 according to Table 2, or    b) a homologue of the molecule or molecular complex, wherein the homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 Å.    
     
     
         22 . A method for identifying a potential modulator of molecule or molecular complex comprising αVβ3 integrin-like binding pocket, the method comprising: 
 a) using the atomic coordinates of αVβ3 integrin amino acids αV:Ala215, αV:Asp218; αV:Asp150; αV:Tyr178; β3:Tyr122; β3:Arg214; β3:Asn215; and β3:Arg216 according to Table 2 ±a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 Å, to generate a three-dimensional structural model of a molecule or molecular complex comprising an αVβ3 integrin-like binding pocket;    b) employing the three-dimensional structural model to design or select said potential modulator;    c) synthesizing the potential modulator; and    d) contacting the potential modulator with the molecule or molecular complex to determine the ability of the potential modulator to interact with the molecule or molecular complex.    
     
     
         23 . A method for evaluating the potential of a chemical entity to associate with a molecule or molecular complex comprising a ligand binding pocket of an αVβ3 extracellular domain, the method comprising: 
 a) employing computational means to perform a fitting operation between the chemical entity and a binding pocket defined by the structural coordinates described in Table 1 or Table 2; and    b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket.    
     
     
         24 . A computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by structure coordinates of Table 1 or Table 2 wherein said computer comprises: 
 a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises the structure coordinates of Table 1 or Table 2 amino acids of the αVβ3 extracellular domain;    b) a working memory for storing instructions for processing said machine-readable data;    c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and    d) a display coupled to said central-processing unit for displaying said three-dimensional representation.    
     
     
         25 . A crystal comprising an integrin αVβ3 extracellular domain complexed with a cyclic RGD peptide.  
     
     
         26 . The crystal of  claim 25  wherein the cyclic RGD peptide ligand comprises an amino acid sequence comprising Arg-Gly-Asp-(D-Phe)-(N-methyl-Val).  
     
     
         27 . The crystal of  claim 25  wherein the cyclic RGD peptide ligand comprises an amino acid sequence comprising Arg-Gly-Asp-(D-Phe)-(N-methyl-Val) in the presence of a metal.  
     
     
         28 . The method of  claim 1  wherein the test compound is: (i) computationally assembled molecular fragments; (ii) selected from a small molecule database; or (iii) computationally created by de novo ligand design.